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Journal of Bioscience and Bioengineering Oct 2019In our previous study, we investigated the relationship between protein evolution and stability through the random mutational drift of an esterase from hyperthermophilic...
In our previous study, we investigated the relationship between protein evolution and stability through the random mutational drift of an esterase from hyperthermophilic archaeon Sulfolobus tokodaii. The results revealed that evolvability, which is the appearance frequency of variants with higher activity than the parent protein, correlates with parental stability. This suggests that protein evolution that does not take stability into account does not make sense. Here, we used those data to further evaluate the relationship between activity and stability in random mutations, revealing that the maximum increase in activity due to mutation conflicts with parental stability. That is, many activated variants are produced when parental stability is high, whereas lower stability offers a few excellent variants with much higher activity. Moreover, we used the random mutant library to compute a novel criterion, robustizability (stabilizability), which is the appearance frequency of variants with a higher stability than the parent protein. Robustizability correlates positively with parental activity and negatively with parental stability. The results indicated that the principle of activity-stability trade-off dominates, in even random mutations. We propose its application in protein engineering via directed evolution by stability selection.
Topics: Archaeal Proteins; Enzyme Activation; Esterases; Gene Library; Mutant Proteins; Mutation; Sulfolobus
PubMed: 30987876
DOI: 10.1016/j.jbiosc.2019.03.017 -
Proceedings of the National Academy of... Dec 2019Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the...
Mutations in Cu/Zn superoxide dismutase (Sod1) have been reported in both familial and sporadic amyotrophic lateral sclerosis (ALS). In this study, we investigated the behavior of heteromeric combinations of wild-type (WT) and mutant Sod1 proteins A4V, L38V, G93A, and G93C in human cells. We showed that both WT and mutant Sod1 formed dimers and oligomers, but only mutant Sod1 accumulated in intracellular inclusions. Coexpression of WT and hSod1 mutants resulted in the formation of a larger number of intracellular inclusions per cell than that observed in cells coexpressing WT or mutant hSod1. The number of inclusions was greater in cells expressing A4V hSod1. To eliminate the contribution of endogenous Sod1, and better evaluate the effect of ALS-associated mutant Sod1 expression, we expressed human Sod1 WT and mutants in human cells knocked down for endogenous Sod1 (Sod1-KD), and in yeast cells. Using Sod1-KD cells we found that the WT-A4V heteromers formed higher molecular weight species compared with A4V and WT homomers. Using the yeast model, in conditions of chronological aging, we concluded that cells expressing Sod1 heterodimers showed decreased antioxidant activity, increased oxidative damage, reduced longevity, and oxidative stress-induced mutant Sod1 aggregation. In addition, we also found that ALS-associated Sod1 mutations reduced nuclear localization and, consequently, impaired the antioxidant response, suggesting this change in localization may contribute to disease in familial ALS. Overall, our study provides insight into the molecular underpinnings of ALS and may open avenues for the design of future therapeutic strategies.
Topics: Aging; Amyotrophic Lateral Sclerosis; Gene Expression Regulation; Gene Knockdown Techniques; HEK293 Cells; Humans; Inclusion Bodies; Molecular Weight; Mutant Proteins; Mutation; Saccharomyces cerevisiae; Superoxide Dismutase-1
PubMed: 31796595
DOI: 10.1073/pnas.1902483116 -
Angewandte Chemie (International Ed. in... Nov 2022Compared with conventional closed-shell fluorophores, radical cations provide an opportunity for development of red-to-NIR fluorophores with small sizes and easy...
Compared with conventional closed-shell fluorophores, radical cations provide an opportunity for development of red-to-NIR fluorophores with small sizes and easy preparation. However, most radical cations reported in the literature suffer from poor stability in water solution and are almost non-emissive. To tackle this challenge, we herein develop a deep-red-emissive and water-stable pyrrole radical cation P⋅ -DPA-Zn, which can be easily generated from P-DPA-Zn by air oxidation. The deep-red-emissive P⋅ -DPA-Zn can be used for imaging-guided mitochondria-targeted delivery of Zn into cancer cells to promote mutant p53 proteins degradation and abrogate mutp53-manifested gain of function, including reduced chemotherapy resistance, inhibited cancer cell migration, decreased tumor cell colony and sphere formation. The water-stable and deep-red emissive pyrrole radical cation is thus promising for cancer theranostic applications.
Topics: Humans; Water; Tumor Suppressor Protein p53; Mutant Proteins; Neoplasms; Cations; Pyrroles
PubMed: 36149752
DOI: 10.1002/anie.202212671 -
Nature Mar 2021Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic-has a furin cleavage site (PRRAR) in its spike...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-a new coronavirus that has led to a worldwide pandemic-has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses. To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.
Topics: Amino Acid Sequence; Animals; Antibodies, Neutralizing; COVID-19; Cell Line; Chlorocebus aethiops; Cricetinae; Female; Furin; Humans; Lung Diseases; Male; Mice; Mice, Transgenic; Models, Molecular; Mutant Proteins; Mutation; Proteolysis; SARS-CoV-2; Serine Endopeptidases; Spike Glycoprotein, Coronavirus; Vero Cells; Virus Replication
PubMed: 33494095
DOI: 10.1038/s41586-021-03237-4 -
PloS One 2020Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder,...
Biallelic mutations in ACP5, encoding tartrate-resistant acid phosphatase (TRACP), have recently been identified to cause the inherited immuno-osseous disorder, spondyloenchondrodysplasia (SPENCD). This study was undertaken to characterize the eight reported missense mutations in ACP5 associated with SPENCD on TRACP expression. ACP5 mutant genes were synthesized, transfected into human embryonic kidney (HEK-293) cells and stably expressing cell lines were established. TRACP expression was assessed by cytochemical and immuno-cytochemical staining with a panel of monoclonal antibodies. Analysis of wild (WT) type and eight mutant stable cell lines indicated that all mutants lacked stainable enzyme activity. All ACP5 mutant constructs were translated into intact proteins by HEK-293 cells. The mutant TRACP proteins displayed variable immune reactivity patterns, and all drastically reduced enzymatic activity, revealing that there is no gross inhibition of TRACP biosynthesis by the mutations. But they likely interfere with folding thereby impairing enzyme function. TRACP exists as two isoforms. TRACP 5a is a less active monomeric enzyme (35kD), with the intact loop peptide and TRACP 5b is proteolytically cleaved highly active enzyme encompassing two subunits (23 kD and 16 kD) held together by disulfide bonds. None of the mutant proteins were proteolytically processed into isoform 5b intracellularly, and only three mutants were secreted in significant amounts into the culture medium as intact isoform 5a-like proteins. Analysis of antibody reactivity patterns revealed that T89I and M264K mutant proteins retained some native conformation, whereas all others were in "denatured" or "unfolded" forms. Western blot analysis with intracellular and secreted TRACP proteins also revealed similar observations indicating that mutant T89I is amply secreted as inactive protein. All mutant proteins were attacked by Endo-H sensitive glycans and none could be activated by proteolytic cleavage in vitro. In conclusion, determining the structure-function relationship of the SPENCD mutations in TRACP will expand our understanding of basic mechanisms underlying immune responsiveness and its involvement in dysregulated bone metabolism.
Topics: Amino Acid Substitution; Autoimmune Diseases; Glycosylation; Humans; Mutant Proteins; Mutation, Missense; Osteochondrodysplasias; Proteolysis; Tartrate-Resistant Acid Phosphatase
PubMed: 32214327
DOI: 10.1371/journal.pone.0230052 -
Cells Nov 2021Orai channels belong to the calcium release-activated calcium (CRAC) channel family. Orai channels are responsible for the influx of extracellular Ca that is triggered... (Review)
Review
Orai channels belong to the calcium release-activated calcium (CRAC) channel family. Orai channels are responsible for the influx of extracellular Ca that is triggered by Ca depletion from the endoplasmic reticulum (ER); this function is essential for many types of non-excitable cells. Extensive structural and functional studies have advanced the knowledge of the molecular mechanism by which Orai channels are activated. However, the gating mechanism that allows Ca permeation through Orai channels is less well explained. Here, we reviewed and summarized the existing structural studies of Orai channels. We detailed the structural features of Orai channels, described structural comparisons of their closed and open states, and finally proposed a "push-pull" model of Ca permeation.
Topics: Animals; Calcium; Calcium Release Activated Calcium Channels; Humans; Ion Channel Gating; Models, Biological; Mutant Proteins; Permeability
PubMed: 34831285
DOI: 10.3390/cells10113062 -
Nucleic Acids Research Jan 2021ProThermDB is an updated version of the thermodynamic database for proteins and mutants (ProTherm), which has ∼31 500 data on protein stability, an increase of 84%...
ProThermDB is an updated version of the thermodynamic database for proteins and mutants (ProTherm), which has ∼31 500 data on protein stability, an increase of 84% from the previous version. It contains several thermodynamic parameters such as melting temperature, free energy obtained with thermal and denaturant denaturation, enthalpy change and heat capacity change along with experimental methods and conditions, sequence, structure and literature information. Besides, the current version of the database includes about 120 000 thermodynamic data obtained for different organisms and cell lines, which are determined by recent high throughput proteomics techniques using whole-cell approaches. In addition, we provided a graphical interface for visualization of mutations at sequence and structure levels. ProThermDB is cross-linked with other relevant databases, PDB, UniProt, PubMed etc. It is freely available at https://web.iitm.ac.in/bioinfo2/prothermdb/index.html without any login requirements. It is implemented in Python, HTML and JavaScript, and supports the latest versions of major browsers, such as Firefox, Chrome and Safari.
Topics: Databases, Protein; Information Storage and Retrieval; Mutant Proteins; Proteins; Statistics as Topic; Thermodynamics
PubMed: 33196841
DOI: 10.1093/nar/gkaa1035 -
Cells May 2020Brain-derived neurotrophic factor (BDNF), a major focus for regenerative therapeutics, has been lauded for its pro-survival characteristics and involvement in both... (Review)
Review
Brain-derived neurotrophic factor (BDNF), a major focus for regenerative therapeutics, has been lauded for its pro-survival characteristics and involvement in both development and recovery of function within the central nervous system (CNS). However, studies of tyrosine receptor kinase B (TrkB), a major receptor for BDNF, indicate that certain effects of the TrkB receptor in response to disease or injury may be maladaptive. More specifically, imbalance among TrkB receptor isoforms appears to contribute to aberrant signaling and hyperpathic pain. A truncated isoform of the receptor, TrkB.T1, lacks the intracellular kinase domain of the full length receptor and is up-regulated in multiple CNS injury models. Such up-regulation is associated with hyperpathic pain, and TrkB.T1 inhibition reduces neuropathic pain in various experimental paradigms. Deletion of TrkB.T1 also limits astrocyte changes in vitro, including proliferation, migration, and activation. Mechanistically, TrkB.T1 is believed to act through release of intracellular calcium in astrocytes, as well as through interactions with neurotrophins, leading to cell cycle activation. Together, these studies support a potential role for astrocytic TrkB.T1 in hyperpathic pain and suggest that targeted strategies directed at this receptor may have therapeutic potential.
Topics: Animals; Brain-Derived Neurotrophic Factor; Embryonic Development; Humans; Mutant Proteins; Neuralgia; Receptor, trkB; Signal Transduction
PubMed: 32403409
DOI: 10.3390/cells9051194 -
Database : the Journal of Biological... Nov 2021Protein domains are functional and structural units of proteins. They are responsible for a particular function that contributes to protein's overall role. Because of...
Protein domains are functional and structural units of proteins. They are responsible for a particular function that contributes to protein's overall role. Because of this essential role, the majority of the genetic variants occur in the domains. In this study, the somatic mutations across 21 cancer types were mapped to the individual protein domains. To map the mutations to the domains, we employed the whole human proteome to predict the domains in each protein sequence and recognized about 149 668 domains. A novel Perl-API program was developed to convert the protein domain positions into genomic positions, and users can freely access them through GitHub. We determined the distribution of protein domains across 23 chromosomes with the help of these genomic positions. Interestingly, chromosome 19 has more number of protein domains in comparison with other chromosomes. Then, we mapped the cancer mutations to all the protein domains. Around 46-65% of mutations were mapped to their corresponding protein domains, and significantly mutated domains for all the cancer types were determined using the local false discovery ratio (locfdr). The chromosome positions for all the protein domains can be verified using the cross-reference ensemble database. Database URL: https://dcmp.vit.ac.in/.
Topics: Deoxycytidine Monophosphate; Humans; Mutant Proteins; Neoplasms; Protein Domains; Proteome
PubMed: 34791106
DOI: 10.1093/database/baab066 -
BMC Bioinformatics Jul 2023Protein engineering aims to improve the functional properties of existing proteins to meet people's needs. Current deep learning-based models have captured evolutionary,...
BACKGROUND
Protein engineering aims to improve the functional properties of existing proteins to meet people's needs. Current deep learning-based models have captured evolutionary, functional, and biochemical features contained in amino acid sequences. However, the existing generative models need to be improved when capturing the relationship between amino acid sites on longer sequences. At the same time, the distribution of protein sequences in the homologous family has a specific positional relationship in the latent space. We want to use this relationship to search for new variants directly from the vicinity of better-performing varieties.
RESULTS
To improve the representation learning ability of the model for longer sequences and the similarity between the generated sequences and the original sequences, we propose a temporal variational autoencoder (T-VAE) model. T-VAE consists of an encoder and a decoder. The encoder expands the receptive field of neurons in the network structure by dilated causal convolution, thereby improving the encoding representation ability of longer sequences. The decoder decodes the sampled data into variants closely resembling the original sequence.
CONCLUSION
Compared to other models, the person correlation coefficient between the predicted values of protein fitness obtained by T-VAE and the truth values was higher, and the mean absolute deviation was lower. In addition, the T-VAE model has a better representation learning ability for longer sequences when comparing the encoding of protein sequences of different lengths. These results show that our model has more advantages in representation learning for longer sequences. To verify the model's generative effect, we also calculate the sequence identity between the generated data and the input data. The sequence identity obtained by T-VAE improved by 12.9% compared to the baseline model.
Topics: Humans; Mutant Proteins; Amino Acid Sequence; Amino Acids; Biological Evolution; Learning
PubMed: 37480001
DOI: 10.1186/s12859-023-05415-9